Ltq velos orbitrap elite

Forty-eight hours after transfection, cells were lysed and equal amounts of extract were precleared using Protein G-agarose beads (Sigma). Extracts were incubated separately with anti-FLAG affinity gel overnight at 4 °C. After washing the immunoprecipitates, bound protein complexes were eluted using 0.15% trifluoroacetic acid (TFA), reduced using 45 mM dithiothreitol and incubated with 1 μg TPCK trypsin overnight. Trypsinized peptides were acidified with 20% TFA, purified using C-18 spin columns (Pierce, IL, USA) and vacuum concentrated. The samples were loaded onto a 75 μm ID X 50 cm (2 μm C18) analytical column (EASY-Spray, Thermo-Fisher Scientific, Odense Denmark). The peptides were eluted over 2 hours at 250 nl/minute using a 0 to 35% acetonitrile gradient in 0.1% formic acid using an EASY nLC 1000 nano-chromatography pump (Thermo-Fisher Scientific, Odense Denmark). The peptides were eluted into a LTQ Velos-Orbitrap Elite hybrid mass spectrometer (Thermo-Fisher, Bremen, Germany) operated in a data dependant mode. MS was acquired at 240,000 FWHM resolution in the FTMS (using a target value of 5 × 10⁵ ions) and MS/MS was carried out in the linear ion trap. 10 MS/MS scans were obtained per MS cycle using a target of 1 × 10⁴ ions and a maximum injection time of 50 ms, and all ions passing the monoisotopic precursor selection (MIPS) filter were fragmented.

A selected subset of samples was analysed using a LTQ-Velos Orbitrap Elite coupled to a Surveyor (both Thermo, Hemel Hampstead, UK) LC system for lipid identification. Ionization was achieved using ion max electrospray source (voltage 4.0 Kv, capillary temperature 275 °C, sheath gas 20 arbitrary (arb) units, aux gas 5 arb, sweep gas 10 arb). For each sample, 10 μl was injected onto a Hypersil Gold C18 (50 × 2.1 mm, 1.9 μm particle size) column and lipids were eluted with a gradient of MeOH/H₂O and MeOH/IPA (Bird et al. 2011 (link)). The LTQ-Orbitrap Velos was controlled either manually or set-up using the data-dependent precursor selection. Generally the selected masses were isolated with a 1.5 m/z width in the linear ion trap and then fragmented using either linear ion trap with 35 % relative collision energy or in the higher-energy collision-induced dissociation collision cell, with a range of collision energies from 5 to 75 % relative collision energy. All spectra were recorded in the Orbitrap set at 100,000 resolution.